体内遗传操作是机械理解疾病和生理过程中基因功能的关键方法。为了促进这一点,已经开发了流行的动物模型如小鼠的胚胎转基因。与更长的时间相比,昂贵的转基因方法,病毒载体,如腺相关病毒(AAV),由于其相对较低的成本和易于生产,作为一种生物工具,转化为整体更大的多功能性。在这篇文章中,我们描述了用于体内有效转导的AAV生产和纯化的方案。重要的是,我们的方法在简化的应用上与其他方法不同,更具成本效益的方法。从这个方法,多达2×1013个含基因组的病毒颗粒(vp),或200个单位,可以在3到4周内生产,用品和试剂的最低成本为1800美元至2000美元,每周人员时间<15小时。这里的单位定义为1×1011vp,我们每只动物的AAV标准剂量,通过尾静脉注射。因此,我们的方法提供了能够转导多达200只动物的量的AAV的生产和纯化。©2023作者。WileyPeriodicalsLLC出版的当前协议。基本方案1:AAV生产基本方案2:AAV纯化。
Genetic manipulation in vivo is a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice has been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno-associated virus (AAV), have grown increasingly in popularity due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this article, we describe protocols for AAV production and purification for efficient transduction in vivo. Importantly, our method differs from others in application of a streamlined, more cost-effective approach. From this method, as many as 2 × 1013 genome-containing viral particles (vp), or 200 units, can be produced within 3 to 4 weeks, with a minimal cost of $1800 to $2000 for supplies and reagents and <15 hr of personnel time per week. A unit here is defined as 1 × 1011 vp, our standard dose of AAV per animal, injected via tail vein. Therefore, our method provides production and purification of AAV in quantities capable of transducing up to 200 animals. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AAV production Basic Protocol 2: AAV purification.